Exploring functional metabolites in preterm infants

Metabolomics is the study of small molecules that represent the functional end points of cellular reactions that can impact health. Necrotising enterocolitis (NEC) and late onset sepsis (LOS) are the main cause of death in preterm infants surviving the initial days of life.

signs of NEC are indistinct, but they may show as a medley of the following medical anomalies: decreased oxygenated blood levels, slowed heart rates, apnoea, temperature fluctuations, parental and tropic feed intolerance, vomiting, bloody stools, abdominal distention and/or discolouration and therefore often infant fatigue. 2 Late onset sepsis (LOS) is neonatal sepsis that occurs in infants later than 72 h after birth. 5 The gold standard method of diagnosis is blood culture, where the causative organism is isolated. However, this can lead to false negatives. Gram-positive organisms are primarily isolated as the causative pathogens for LOS in infants, 6 largely resulting from Coagulase-negative staphylococci (CoNS) (54%) and, to a lesser extent, Enterobacteriaceae (Gram negative, 21%) and Staphylococcus aureus (Gram positive, 18%). 7 The most common cause of death in premature infants is NEC and LOS. 4 Twenty-one per cent of all deaths in infants born 24-31 weeks gestation are caused by NEC and/or infection. 8 One in 10 infants are born preterm, and this number is increasing every year. 9 Preterm births is a major cause of death and long-term morbidity, including increased risk of cerebral palsy and costs for surgery for NEC and intensive care requirements, costing the public sector approximately £3 billion per year in the United Kingdom. 10,11

| What causes NEC and LOS
Necrotising enterocolitis can arise from several different risk factors, including abnormal bacterial colonisation, bacterial translocation and activation of the cytokine cascade, decrease in epidermal growth factor, increase in platelet activating factor and mucosal damage from free radical production. 2 Infants who show intolerance to enteral feeding are likely to have higher parenterally administered calories, which has also been linked to a higher risk of developing NEC. 12 Furthermore, a relationship between vitamin deficiency and the potential benefit of supplementation of vitamin D in pregnancy is thought to be important in preventing NEC in preterm infants. 13 There are a number of risk factors for developing NEC that include low birth weight, prematurity and formula rather than breastfeeding. 14 The impact of antibiotics may predispose the neonate to NEC by delaying the colonisation of potentially beneficial bacteria and reducing the diversity of the intestinal microbiome. 15 For instance, a correlation between antibiotic duration and risk of developing NEC among infants without culture-proven sepsis has been reported. 16 Early onset sepsis, defined as sepsis onset within 72 h of birth, most likely occurs during birth, with group B Streptococcus (58%) and Escherichia coli (18%) from the mothers' genital tract is leading to subsequent infection. 7,17 LOS, on the other hand, is likely to be hospital acquired. As survival has improved in extreme preterm infants, so too has the length of time the preterm infants may spend in intensive care. This increases the use of central venous lines, parental nutrition, mechanical ventilation, use of catheters, endotracheal tubes and other invasive procedures which likely increase rates of LOS. 17,18

| Current best practice treatment options for NEC and LOS
Current NEC treatment practices include bowel decompression, antibacterial therapy and haematological and electrolyte support. 2 The provision of breast milk, trophic feeds and the advancement of enteric feeds can reduce the risk of NEC. 2 Further preventative strategies include the use of antibacterial, antioxidants, epidermal growth factor and probiotics. 2 Prompt antibiotic treatment is needed for infants with LOS as infants can deteriorate rapidly. 19 Diagnostic blood culture is a timely process and can produce false negatives due to the small blood samples taken from the preterm neonates. 20 LOS is treated with an array of different antibiotic types which are typically commenced while awaiting results, which may contribute to immunosuppression and antibiotic resistance for those infants, 21 but it is necessary to control the infection.

| ME TABOLITE S
The human gut contains a vast collection of metabolically active microbial organisms and a diverse array of biological molecules and cells which make up the human gut environment. 22 This human gut environment also includes a vast number of immune cell types including T cells, phagocytes, lymphocytes, stroma cells and molecules including proteins, sugars and lipids, as well as the microbiome and metabolome. The gut microbiome is a microbial community that includes bacteria, bacteriophages, viruses, archaea and fungi. The metabolome is the collection of functional low molecular weight compounds (metabolites) that represents the functional end points of cellular reactions in a biological system, including carbohydrates, fatty acids, hormones and amino acids. Metabolomics is the study of small molecules, and metabolomic research aims to characterise metabolites produced by gut microbes and host cells 23  • Preliminary studies investigating preterm infants have found selected metabolites are associated with disease, but further observational and experimental research is needed.
junction integrity within the gut) are produced by anaerobic gut bacteria including bifidobacteria. Metabolites that are observed through metabolomics are impacted by an array of variables such as lifestyle (nutritional and environmental), drug substance usage, disease, genetics 24 and non-biological factors. Metabolomics is a developing technology that has the potential to inform the practice of precision medicine due to its ability to profile such large numbers of metabolites, providing widespread insights into biological processes. 26

| Sample types
Metabolites can be found in human cells and a range of biological material including urine, blood and stool, providing multiple sample options for metabolomic research. Blood (including serum and plasma) has been the preferred sample type for metabolite work to date ( Figure 1). 27 Nonetheless, studies involving urine and stool are increasing, and there is a general rise in metabolite studies over the past ~10 years.
Each biological sample choice has a unique biochemical composition which is determined by response to physiological stimuli, resulting in metabolic activity and metabolome profiles that are unique and specific to an individual. 25 Metabolomic analysis of blood (or serum or plasma) provides a snapshot of absorbed luminal metabolites and host metabolic activity across the entire human body. 28 Notably, preterm infants have limited circulating blood volumes and are at higher risk of developing sepsis; thus, invasive procedures are generally not permissible for solely research purposes in this population. For this reason, researchers have turned to salvaging samples that are collected for clinical procedures and where remaining sample would otherwise be discarded. 29 This can limit the utility of the sample, where volume will be limited and samples may have been collected and stored in nonoptimal conditions for downstream metabolomic experiments. Stool as a sample option in comparison with blood/serum/plasma provides a non-invasive means of measuring functional small molecules derived from the gut lumen, including from microbes that colonise in the gut. Stool is a more direct sample obtained from the site of medical interest for NEC. Urine is a downstream sample in comparison and includes more water soluble and metabolic by products. 28 Because urine contains concentrated waste products of many metabolic processes that give insight into the underlying metabolic activity, it is possible to analyse a large number of compounds. 25,30 For neonatal practice, non-invasive techniques are used to collect the urine samples, where a sterile ball of cotton is placed into the nappy/diaper and absorbed urine is transferred with a sterile syringe into a sterile cryovial. 10,25 Stool sample collection in neonatal practice uses sterile sampling spoons to directly transfer the sample from the infants nappy/diaper into sterile sample containers. 10 Where immediate analysis is not possible, samples are typically frozen at −80°C for long-term storage. Non-biological factors such as sample collection, transport, storage temperature, duration of storage and freeze-thaw cycles are known to impact the metabolomic output. 31 This highlights the importance of standardising any sample handling practices before analysis so that the metabolic integrity is preserved. 31 Although optimal sample collection and storage conditions are not always possible during clinical studies, it is usually appropriate to utilise samples that have been collected, stored and processed in the same way.

| Microbiome and metabolome interplay
Most often the microbiome is studied for taxonomic purposes, to identify what bacteria are present within the human gut. At the taxonomic level, the gut microbiome is known to differ between individuals, yet many metabolic pathways are shared, suggesting there are multiple ways that different microbes may produce or modify the same metabolites. 24 The microbiome and the metabo-  NEC preterm infants Urine metabolomic output showed a high urinary alanine:histidine ratio that could predict NEC.
This study couples microbiome and metabolomic analysis but was small in cohort size and only included a small number of NEC infants. for blood samples, with 1,054 randomly selected stool samples for metagenomic sequencing. 22 The study showed 82% of metabolic pathways were shared between individuals, in comparison with bacterial species in common identified at a lesser percentage of 43%. 22 The study followed this with estimations that the microbiome was involved in metabolite microbiome interplay for 71% of faecal and 15% blood metabolites, demonstrating stool metabolic content are strongly linked. 22  Studies are now producing evidence, with the use of stool samples, of microbiome-metabolome linkage. 22,32 Such work demonstrates feasibility for multi-omic study design leading to identification of metabolites of interest that could be used as biomarkers of disease in preterm infants.

| Metabolites and human health
The large number of different bacteria in the gut microbiome generates compounds that are part of the metabolic signalling network and can influence human health. The use of metabolites for disease diagnosis has been used for decades, for example, measuring phenylalanine in neonates to screen for phenylketonuria. 33 It has also already been identified that microbially produced metabolites can impact immunoregulation. 34  NEC preterm infantsl -arginine concentrations (the substrate for nitric oxide synthesis) Infants with NEC at the time of diagnosis had lower arginine concentrations in their blood plasma analysed in comparison with healthy infants.
The study only included a small number of NEC infants within the cohort.

TA B L E 1 (Continued)
cytokines. 34 They can also act on antigen presenting cells in the brain or lungs which leads to a decreased inflammatory response. 34 Although the phases of maturation of metabolites from neonate to adult are not as clearly defined as the microbiome, 35  onates. 37 They found that urinary metabolites were fewer in number and less diverse in neonates compared with children or adults. 37 Interestingly, neonates had higher levels of particular amino acids including collagen-associated amino acids and acylcarnitines, which are needed to support rapid growth early in life. 37 Other work has  39 The majority of the samples taken within this study were taken on the second day after the infants birth. 39 Comparisons were made between Day 1 and 2 of life, which did not show evidence of metabolomic change; however, there were limited samples taken on Day 1, so this finding is inconclusive. 39 This study did highlight variations between preterm and term infants observed in NMR spectral output, partial least squares-discriminant analysis (PLS-DA) statistical assessment, and 25 identical metabolite changes were visible in comparison Volcano plot analysis. 39 There were gender ratio inconsistencies, however, between the preterm and term control groups, so perhaps, a more balanced cohort group in future studies could help verify these findings. 39

| Techniques for measuring metabolites
No one analytical technique has been assigned as the gold standard for metabolomics, and each technique has benefits and limitations. 40 Selection also considers if the study is for targeted or untargeted metabolite exploration. With predefined targeted metabolites of interest, targeted mass spectrometry (MS) is typically used.
Targeted MS has high sensitivity, selectivity, accuracy and precision.
When the target metabolites or pathways are unknown or when a completely unbiased approach is preferred, liquid chromatography mass spectrometry (LC-MS) or nuclear magnetic resonance is typically employed. were shown in infants who were extremely preterm and/or went on to develop NEC. 12 Metabolic differences were also identified in infants prior to NEC onset and showed links to nutrients administered, feeding intolerances and calorie intake prior to NEC onset. 12 On the first day of life, several metabolites and metabolite ratios were identified in infants that went on to develop NEC including alanine, phenylalanine, arginine and citrulline/phenylalanine. 12  Despite the differing cohort characteristics, sizes, sample types and analytical methods, several metabolites were consistently found to differ in abundance in NEC infants compared with controls, including alanine, arginine, proline, glutamine, histamine, creatinine and betaine. 12,[43][44][45]48,49 Alanine, arginine, proline and glutamine are amino acids that are building blocks of proteins used in the body as a source of energy for muscles and the central nervous system.

| NEC AND LOS A SSO CIATED ME TABOLITE S
Glutamine has been specifically used in supplementation studies in the past to help treat immunosuppressed individuals. 50 Histamine is a chemical that is also connected to immunity, and it is thought that histamine intolerance is a disorder connected to gut health. 51 Arginine has been reviewed in previous literature as arginine is a substrate for nitric oxide synthesis. 12 It has been speculated that nitric oxide synthesis is connected to NEC; however, this remains uncertain. 12 To summarise, there are minimum metabolite studies looking at urine samples or combining multiple sample options within the same study (Table 1). Differing biofluids provide their own metabolic 'fingerprint', 25  Existing studies have reported associations between the metabolite profiles of serum, urine and stool in health and disease in preterm infants. Some studies have found selected metabolites are associated with disease; however, additional or larger studies would be required to validate. Furthermore, recent work has begun to untangle how microbially produced metabolites can impact immunoregulation of the infant. Although metabolic exploration in infants is still an underutilised technology, there is clearly huge potential for this field in developing novel biomarkers for early detection of NEC and LOS in preterm infants, which could reduce unnecessarily treating or not treating preterm infants accurately.

C. J. S. declares receiving lecture honoraria from Danone Early Life
Nutrition and Nestle Nutrition Institute but has no share options or other conflicts.